Despite the existence of an effective quadrivalent vaccine, targeting the high risk human papillomavirus (HPV) serotypes 6, 11, 16 and 18, the incidence of HPV-induced cancers in the USA remains high and the incidence of HPV-positive head and neck and anal cancers, which are primarily caused by HPV-16, is actually increasing. It is well established that the maintenance of the transformed state in HPV-induced cancers is dependent on the continued expression of two viral oncogenes, E6 and E7, that target and inactivate the cellular tumor suppressors p53 and Rb, respectively. Recently, we demonstrated that the bacterial CRISPR/Cas antiviral adaptive immune system from Streptococcus pyogenes (Spy) could be repurposed to effectively target and inactivate either the HPV E6 or E7 gene in cultured HPV-transformed cells. Expression of Spy Cas9 and HPV-specific single guide RNAs (sgRNAs) resulted in the mutational inactivation of E6 or E7 and in the induction of p53 or Rb activity, respectively, leading to cell cycle arrest and eventual cell death. In this application, e wish to extend these studies by demonstrating that Cas9/sgRNA combinations delivered to HPV-16-transformed, patient-derived tumors, explanted into immunodeficient mice, using adeno-associated virus (AAV) vectors, can effectively and specifically shrink these tumors. For this purpose, we have identified and characterized a novel Cas9 gene, derived from Staphylococcus aureus (Sau), that is as active as Spy Cas9 in gene editing yet sufficiently small, at ~3.2 kb, to fit into an AAV vector along with two HPV-specific sgRNAs and relevant transcriptional regulatory elements. AAV vectors expressing HPV-16-specific Sau Cas9/sgRNA combinations will first be tested for effectiveness in culture, using the HPV-16 transformed SiHa cell line, then injected into mice carrying several different HPV-16-transformed, patient derived tumor xenografts and monitored for tumor size, growth and viability. We hypothesize that this approach will prove able to effectively eliminate HPV-16-dependent tumors in this preclinical murine model system, thus providing proof-of-principle that a similar approach could work not only as a clinical approach to the treatment of HPV-16-induced human tumors but also possibly as a strategy to treat any cancer that is entirely dependent on the continued functional expression of a specific pro-oncogenic gene of viral or non-viral origin.